Heterobifunctional polymeric bioconjugates

ABSTRACT

Heterobifunctional polymeric prodrug platforms for delivering biologically active compounds, including proteins, monoclonal antibodies and the like are disclosed. One preferred compound is 
                         
Methods of making and using the compounds and conjugates described herein are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 10/394,393 filed Mar. 21, 2003, the contents of which areincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the synthesis of high molecular weightheterobifunctional polymeric conjugates useful in the targeting anddelivery of therapeutic agents. Methods of making and using theconjugates are also disclosed.

BACKGROUND OF THE INVENTION

Targeting and drug delivery of therapeutics is becoming increasinglyimportant especially with the use of cytotoxics in the treatment ofcancer. A number of methods have been used to selectively target tumorswith therapeutic agents to treat cancers in humans and other animals.Targeting moieties such as monoclonal antibodies (mAb) or theirfragments have been conjugated to linear polymers via their side chainfunctional groups. However, this approach usually results in reducedreceptor binding affinity either due to changes in the chemicalproperties of the antibodies or due to folded configuration of polymersthat imbed the targeting moiety in the random coiled structure. Ideally,a new conjugate would encompass both a targeting functionality as wellas a therapeutic value.

Recently, heterobifunctional polymeric conjugates having a targetingfunctional group on one end and a therapeutic moiety (e.g. achemotherapeutic drug) on the opposite end has been disclosed, see USPatent Application 2002/0197261A1. The polymer conjugates employed havea polymeric spacer bonded to a polymeric carrier containing multipleside-chain functional groups that allow the attachment of multiple drug,molecules (e.g. poly(1-glutamic acid)) on one end, with the other end ofthe polymeric spacer bonded to a targeting moiety. However, themolecular weight of the polymeric spacer portion is considerably low.

Methods of preparing higher molecular weight heterobifunctional polymerconstructs have been disclosed, see US Patent Application2002/0072573A1. However, these methods involve the polymerization ofmonomers which in itself is not ideal due to undesirable polymerdispersity. Other previous methods have involved anionic ethoxylationand difficult purification steps. Attempting to achieve high molecularweight polymer substrates using the techniques above has resulted inpoor quality and poor yield of desired product.

Due to the inadequacies of the present methods there exists a need forimproved methods of making high molecular weight heterobifunctionalpolymer substrates that produce high yield and high purity substrates atthe same time retaining low polymer dispersity. It would also bedesirable to provide compounds incorporating heterobifunctional polymersubstrates as a means of targeting and delivering therapeutically activecompounds. The present invention addresses these needs.

SUMMARY OF THE INVENTION

In one aspect of the invention there are provided compounds of theformula (I):

wherein:

X₁-X₆ are independently O, S or NR₁;

R₄₄ and R_(44′) are independently selected polyalkylene oxides;

R₁ is selected from among hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls,C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, aralkyls, and C₃₋₈substituted cycloalkyls;

R₄₀₋₄₃ are independently selected from among hydrogen, C₁₋₆ alkyls,C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy, phenoxy andC₁₋₆ heteroalkoxy;

y, and y′ are independently zero or a positive integer;

p and p′ are independently zero or one;

n and n′ are independently one or a positive integer;

a and b are independently zero or a positive integer, provided that a+bis greater than or equal to two;

z is 1 or a positive integer;

D₁ and D₂ are independently selected from among B, leaving groups,activating groups, OH and terminal groups; and

B is selected from among biologically active moieties, diagnostic agentsand OH.

In a preferred embodiment, X₁-X₆ are independently Q or NR₁, R₁ ishydrogen, a and b are independently selected integers from 1 to about20, y and y′ are independently 0, 1 or 2, p and p′ are each 1, D₁ and D₂are independently selected from among leaving groups and terminal groupsand B, wherein B is a biologically active moiety such as, a drug, anamino or hydroxyl-containing residue, a diagnostic agent such as a dye,chelating agent or isotope labeled compound, a leaving group oractivating group.

For purposes of die present invention, the term “residue” shall beunderstood to mean that portion of a biologically active compound whichremains after it has undergone a substitution reaction in which theprodrug carrier has been attached.

For purposes of the present invention, the term “alkyl” shall beunderstood to include straight, branched, substituted C₁₋₁₂ alkyls, C₃₋₈cycloalkyls or substituted cycloalkyls, etc.

Some of the chief advantages of the present invention include novel highmolecular weight heterobifunctional polymeric conjugates capable ofenhancing the circulating half-life and solubility of native orunmodified molecules as well as methods of building such conjugateswherein high purity is maintained without needing a chromatography step.Another advantage of the methods of the present invention is theretention of low polymer dispersion with increasing molecular weight ofthe polymer conjugates. A further advantage of the present invention isthat it allows for the artisan to design a drug conjugate that can havethe same or different groups on either side of the polymeric portion.This advantage allows the artisan to tailor a compound to contain adelivery or targeting functionality and a therapeutic functionalitywithin the same conjugate depending on a particular need.

Methods of making and using the compounds and conjugates describedherein are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 through 9 schematically illustrate methods of forming compoundsof the present invention which are described in the Examples.

DETAILED DESCRIPTION OF THE INVENTION

A. Formula (I)

In one aspect of the invention there are provided compounds of theformula (I):

wherein:

X₁-X₆ are independently O, S or NR₁;

R₄₄ and R_(44′) are independently selected polyalkylene oxides;

R₁ is selected from among hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls,C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, aralkyls, and C₃₋₈substituted cycloalkyls;

R₄₀₋₄₃ are independently selected from among hydrogen, C₁₋₆ alkyls,C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy, phenoxy andC₁₋₆ heteroalkoxy;

y, and y′ are independently zero or a positive integer;

p and p′ are independently zero or one;

n and n′ are independently one or a positive integer;

a and b are independently zero or a positive integer, provided that a+bis greater than or equal to two;

z is 1 or a positive integer;

D₁ and D₂ are independently selected from among B, leaving groups,activating groups, OH and terminal groups; and

B is selected from among biologically active moieties, diagnostic agentsand OH.

In a preferred embodiment of the compound of formula (I):

X₁-X₆ are independently O or NR₁;

R₁ is selected from among hydrogen, C₁₋₆ alkyls, C₁₋₆ heteroalkyls,aralkyls, and C₁₋₆ substituted alkyls;

y, and y′ are independently 0 or an integer between 1 and 18;

p and p′ are independently 0 or 1;

n and n′ are independently selected integers between 0 and 100;

a and b are independently selected integers between 1 and 20; and

z is a positive integer.

More preferably,

X₁-X₄ are independently NR₁;

X₅-X₆ are each O;

R₄₄ and R_(44′) are each —(CH₂—CH₂—O)—;

R₁ is hydrogen or methyl;

y and y′ are each 0, 1 or 2;

p and p′ are each 1;

n and n′ are independently selected integers between 70 and 80;

a and b are independently selected integers between 5 and 10;

z is a positive integer;

D₁ and D₂ are independently selected from among OH, halogens, targetingagents, drugs, enzymes, proteins, therapeutically active compounds,dyes, chelating agents and isotope labeled compounds.

In yet another preferred embodiment of a compound of formula (I), D₁ andD₂ are independently selected terminal groups such as:

wherein:

Y₁₋₆ are independently O or NR_(1′);

R_(1′) is hydrogen or methyl;

R₂₋₈ are independently selected from among hydrogen and C₁₋₆ alkyls;

Ar is a moiety which forms a multi-substituted aromatic hydrocarbon or amulti-substituted heterocyclic group;

L₁₋₂ are independently selected bifunctional linkers;

e and f′ are each one;

c, c′ and e′ are independently zero or one;

d, f and d′ are independently zero or one; and

B′ is selected from among leaving groups, activating groups, OH,biologically active moieties and diagnostic agents.

In another preferred aspect of the invention, there are provided polymerconjugates of the formula (Ia):

wherein:

Y₇₋₉ are independently O or NR_(1″);

R_(1″) is hydrogen or methyl;

R₉₋₁₈ are independently hydrogen or C₁₋₆ alkyls;

L₃₋₄ are independently selected bifunctional linkers;

Q is selected from among moieties actively transported into a targetcell, hydrophobic moieties, bifunctional linking moieties andcombinations thereof;

l, k, m and o are independently positive integers;

j and h are independently zero or one;

g, and i are each one;

q is zero or one;

B′ is selected from among leaving groups, activating groups, OH,biologically active moieties and diagnostic agents;

D₁₀ and D₁₁ are selected from the same group which defines D₁ ortogether form a terminal group of the formula:

In yet another preferred aspect of the invention, D₁ and D₂ areindependently selected terminal groups such as:

wherein D′ is one of

where B′ is selected from among leaving groups, activating groups, OH,biologically active moieties and diagnostic agents.B. Linker Moieties L₁₋₄

As shown above, the invention may include the bifunctional linkingmoieties L₁-L₄. Preferably, L₁-L₄ are independently selected from amongthe non-limiting list:

wherein:

R₃₄-R₃₈ are independently selected from among hydrogen, C₁₋₆ alkyls,C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈substituted cycloalkyls, aryls, substituted aryls, aralkyls, C₁₋₆heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy, phenoxy andC₁₋₆ heteroalkoxy;

R₃₉ is selected from among hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls,C₃₋₈ cycloalkyls, C₁₋₆ substituted alkyls, C₃₋₈ substituted cycloalkyls,aryls, substituted aryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆heteroalkyls, C₁₋₆ alkoxy, phenoxy, C₁₋₆ heteroalkoxy, NO₂, haloalkyland halogens;

a′ and b′ are independently selected positive integers.

C. Description of the Ar Moiety

In certain aspects of the invention, it can be seen that the Ar moietyis a moiety which when included in Formula (I) forms a multi-substitutedaromatic hydrocarbon or a multi-substituted heterocyclic group. A keyfeature is that the Ar moiety is aromatic in nature. Generally, to bearomatic, the −n electrons must be shared within a “cloud” both aboveand below the plane of a cyclic molecule. Furthermore, the number of pelectrons must satisfy the Hückel rule (4n+2). Those of ordinary skillwill realize that a myriad of moieties will satisfy the aromaticrequirement of the moiety for formula (I) and thus are suitable for useherein.

Some particularly preferred aromatic groups include:

wherein R₆₂₋₆₇ are independently selected from the same group whichdefines R₂.

Other preferred aromatic hydrocarbon moieties include, withoutlimitation

wherein Z and E are independently CR₆₈ or NR₆₉; and J is O, S or NR₇₀where R₆₈₋₇₀ are selected from the same group at that which defines R₂or a cyano, nitro, carboxyl, acyl, substituted acyl or carboxyalkyl.Isomers of the five and six-membered rings are also contemplated as wellas benzo- and dibenzo-systems and their related congeners are alsocontemplated. It will also be appreciated by the artisan of ordinaryskill that aromatic rings can optionally be substituted withhetero-atoms such as O, S, NR₁, etc. so long as Hückel's rule is obeyed.Furthermore, the aromatic or heterocyclic structures may optionally besubstituted with halogen(s) and/or side chains as those terms arecommonly understood in the art.D. Polyalkylene Oxides

Referring to Formula (I) it can be seen that R₄₄ is a polymer moietysuch as polyalkylene oxide. Suitable examples of such polymers includepolyethylene glycols which are substantially non-antigenic. Also usefulare polypropylene glycols, such as those described in commonly-assignedU.S. Pat. No. 5,643,575. Other PEG's useful in the methods of theinvention are described in Shearwater Polymers, Inc. catalog“Polyethylene Glycol and Derivatives 2001”. The disclosure of each isincorporated herein by reference.

Although PAO's and PEG's can vary substantially in weight averagemolecular weight, preferably, R₄₄ has a weight average molecular weightof from about 2,000 to about 136,000 Da inmost aspects of the invention.More preferably, R₄₄ has a weight average molecular weight of from about3,400 to about 65,000 Da, with a weight average molecular weight of fromabout 3,400 to about 20,000 Da being most preferred.

The polymeric substances included herein are preferably water-soluble atroom temperature. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof, provided that the water solubility of theblock copolymers is maintained.

E. Formula (I) D₁, D₂ B and B′ Groups

1. Leaving Groups

In those aspects of formula (I) where D₁, D₂ are independently selectedleaving groups, suitable moieties include, without limitation, groupssuch as halogens, activated carbonates such as hydroxysuccinimidylcarbonate, carbonyl imidazole, cyclic imide thiones, isocyanates,N-para-nitrophenol, N-hydroxyphtalimide, N-hydroxybenzotriazolyl,imidazole, tosylates,

Other suitable leaving groups will be apparent to those of ordinaryskill.

For purposes of the present invention, leaving groups are to beunderstood as those groups which are capable of reacting with anucleophile found on the desired target, i.e. a biologically activemoiety, a bifunctional spacer, intermediate, etc. The targets thuscontain a group for displacement, such as NH₂ groups found on proteins,peptides, enzymes, naturally or chemically synthesized therapeuticmolecules such as doxorubicin.

2. Activating Groups

In those aspects of formula (I) where D₁, D₂, B and B′ are independentlyactivating groups. Non-limiting examples of such functional groupsinclude maleimidyl, vinyl, residues of vinylsulfone, hydroxy, amino,carboxy, mercapto, hydrazide, carbazate and the like. Once attached tothe polymer conjugate the functional group, (e.g. maleimide), can beused to attach the polymer conjugate to a target such as the cysteineresidue of a polypeptide, amino acid or peptide spacer, etc.

3. Biologically Active Moieties

In those aspects of formula (I) where D₁, D₂, B or B′ are residues of anamine- or hydroxyl-containing compound. A non-limiting list of suchsuitable compounds include residues of organic compounds, enzymes,proteins, polypeptides, etc. Organic compounds include, withoutlimitation, moieties such as anthracycline compounds includingdaunorubicin, doxorubicin; p-aminoaniline mustard, melphalan, Ara-C(cytosine arabinoside) and related anti-metabolite compounds, e.g.,gemcitabine, etc. Alternatively, the moiety can be a residue of anamine- or hydroxyl-containing cardiovascular agent, anti-neoplasticagent such as camptothecin and paclitaxel, anti-infective, anti-fungalsuch as nystatin, fluconazole and amphotericin B, anti-anxiety agent,gastrointestinal agent, central nervous system-activating agent,analgesic, fertility agent, contraceptive agent, anti-inflammatoryagent, steroidal agent, agent, etc.

In addition to the foregoing, the biologically active moiety can also bea residue of an enzyme, protein, polypeptide, single chain antigenbinding proteins, (SCA's) monoclonal antibodies such as CC49, fragmentsthereof, etc. SCA's of monoclonal antibodies are also contemplated.Suitable proteins include but are not limited to, polypeptides, enzymes,peptides and the like having at least one available group for polymerattachment, e.g. an e-amino, cystinylthio, N-terminal amino, includematerials which have physiological or pharmacological activities as wellas those which are able to catalyze reactions in organic solvents.

Proteins, polypeptides and peptides of interest include, but are notlimited to, hemoglobin, serum proteins such as blood factors includingFactors VII, VIII, and IX; immunoglobulins, cytokines such asinterleukins, i.e. IL-1 through IL-13, etc., α, β and γ interferons,colony stimulating factors including granulocyte colony stimulatingfactors, platelet derived growth factors and phospholipase-activatingprotein (PLAP). Other proteins of general biological or therapeuticinterest include insulin, plant proteins such as lectins and ricins,tumor necrosis factors and related proteins, growth factors such astransforming growth factors, such as TGFα or TGFβ and epidermal growthfactors, hormones, somatomedins, erythropoietin, pigmentary hormones,hypothalamic releasing factors, antidiuretic hormones, prolactin,chorionic gonadotropin, follicle-stimulating hormone,thyroid-stimulating hormone, tissue plasminogen activator, and the like.Immunoglobulins of interest include. IgG, IgE, IgM, IgA, IgD andfragments thereof.

Some proteins such as the interleukins, interferons and colonystimulating factors also exist in non-glycosylated form, usually as aresult of using recombinant techniques. The non-glycosylated versionsare also among the proteins of the present invention.

Enzymes of interest include carbohydrate-specific enzymes; proteolyticenzymes, oxidoreductases, transferases, hydrolases, lyases, isomerasesand ligases. Without being limited to particular enzymes, examples ofenzymes of interest include asparaginase, arginase, arginine deaminase,adenosine deaminase, superoxide dismutase, endotoxinases, catalases,chymotrypsin, lipases, uricases, adenosine diphosphatase, tyrosinasesand bilirubin oxidase. Carbohydrate-specific enzymes of interest includeglucose oxidases, glucodases, galactosidases, glucocerebrosidases,glucouronidases, etc.

Also included herein is any portion of a biological polymerdemonstrating in vivo bioactivity. This includes amino acid sequences,nucleic acids (DNA, RNA), peptide nucleic acids (PNA), antibodyfragments, single chain binding proteins, see, for example U.S. Pat. No.4,946,778, disclosure of which is incorporated herein by reference,binding molecules including fusions of antibodies or fragments,polyclonal antibodies, monoclonal antibodies and catalytic antibodies.

The proteins or portions thereof can be prepared or isolated by usingtechniques known to those of ordinary skill in the art such as tissueculture, extraction from animal sources, or by recombinant DNAmethodologies. Transgenic sources of the proteins, polypeptides, aminoacid sequences and the like are also contemplated. Such materials areobtained from transgenic animals, i.e., mice, pigs, cows, etc., whereinthe proteins are expressed in milk, blood or tissues. Transgenic insectsand baculovirus expression systems are also contemplated as sources.Moreover, mutant versions of proteins, such as mutant interferons arealso within the scope of the invention.

Other proteins of interest are allergen proteins such as ragweed,Antigen E, honeybee venom, mite allergen, and the like. The foregoing isillustrative of the proteins which are suitable for the presentinvention. It is to be understood that those proteins, as definedherein, not specifically mentioned but having an available amino groupare also intended and are within the scope of the present invention.

In a preferred aspect of the invention, the amino- orhydroxyl-containing compound is a biologically active compound that issuitable for medicinal or diagnostic use, in the treatment of animals,e.g., mammals, including humans, for conditions for which such treatmentis desired. The foregoing list is meant to be illustrative and notlimiting for the compounds which can be modified. Those of ordinaryskill will realize that other such compounds/compositions can besimilarly modified without undue experimentation. It is to be understoodthat those biologically active materials not specifically mentioned buthaving suitable attachment groups are also intended and are within thescope of the present invention.

The only limitations on the types of amino- or hydroxyl containingmolecules suitable for inclusion herein is that there is available atleast one (primary or secondary) amine- or hydroxyl- which can react andlink with the polymeric conjugate and that there is not substantial lossof bioactivity after the prodrug system releases and regenerates theparent compound.

4. Diagnostic Agents

In those aspects of formula (I) where D₁, D₂, B and B′ is a diagnosticagent, a non-limiting list of suitable agents includes dyes, chelatingagents, and isotope labeled compounds and other labeling compounds suchas Green Fluorescent Protein (GFP).

F. Q Moieties and their Function

In one aspect of the invention Q is L₅-C(═Y₁₀) wherein L₅ is abifunctional linker selected from among the group which defines L₁, L₂,L₃, and L₄ and Y₁₀ is selected from among the same groups as that whichdefines Y₁₋₉. In this aspect of the invention, the Q group servers asthe linkage between the B′ groups and the remainder of the polymericconjugate.

In other aspects of the invention, Q is a moiety that is activelytransported into a target cell, a hydrophobic moiety, and combinationsthereof. Although Q is preferably monovalent, Q can optionally bebivalent or multivalent so to allow attachment of more than one B′ groupto the polymer conjugate. In order to achieve the active transport, Qcan include an amino acid or peptide residue, a sugar residue, a fattyacid residue, a C₆₋₁₈ alkyl a substituted aryl, a heteroaryl, —C(═O),—C(═S) or —C(═NR₂₈), wherein R₂₈ is H, lower alkyl, etc.

This aspect of the invention is broadly based upon the principle thatbiologically active materials suitable for incorporation into thepolymer conjugates may themselves be substances/compounds which are notactive after hydrolytic release from the polymer substrate, but whichwill become active after undergoing a further chemical process/reaction.With this embodiment, a therapeutic or diagnostic agent, peptide,polypeptide, etc. that is delivered to the bloodstream by the polymersystem, will remain inactive until entering or being activelytransported into a target cell of interest, whereupon it is activated byintracellular chemistry, e.g., by an enzyme or enzyme system present inthat tissue or cell.

The compounds of this aspect of the invention are prepared so that invivo hydrolysis of the polymer-based conjugate cleaves the conjugate soas to release the active biological material (designated B′ herein)into, extracellular fluid, while still linked, to the Q moiety. Thebiologically active materials in this aspect of the invention arepreferably, but not exclusively, small molecule therapeutic and/ordiagnostic agents. For example, one potential Q-B′ combination isleucine-doxarubacin, another is amino acid-linked camptothecin orpaclitaxel and the tissue to be treated is tumor tissue.

Without intending to be bound by any theory or hypothesis as to how theinvention might operate, it is believed that, depending upon theadditional moiety selected as a transport enhancer, the rate oftransport of a biologically active material into tumor cells is by thedelivery of a biologically active material into extracellular tissuepace, e.g., of a tissue exhibiting an EPR effect, in a protected and/ortransport-enhanced form.

In a further still option, the transport enhancer (Q) is selected fromamong known substrates for a cell membrane transport system. Simply byway of example, cells are known to actively transport certain nutrientsand endocrine factors, and the like, and such nutrients, or analogsthereof, are readily employed to enhance active transport of abiologically effective material into target cells. Examples of thesenutrients include amino acid residues, peptides, e.g., short peptidesranging in size from about 2 to about 10 residues or more, simple sugarsand fatty acids, endocrine factors, and the like.

Short peptides are, for example, peptides ranging from 2 to about 10, ormore, amino acid residues, as mentioned supra. In this embodiment of theinvention, it is believed that such peptide transport enhancers need notbe hydrophobic, but are thought to function in other ways to enhanceuptake and/or to protect the linked small molecule agents from prematurehydrolysis in the general bloodstream. For instance, peptide transportenhancers, and other transport enhancers of similar molecular weightranges, are thought to sterically hinder cleavage from the biologicallyactive agent by plasma-based hydrolytic enzymes, but are then cleavedwithin a target cell by various peptides and/or proteases, such ascapthesins.

In certain preferred aspects Q is a hydrophobic moiety. Without meaningto be bound to any theory or hypothesis as to how hydrophobicitycontributes to efficacy, it is believed that a hydrophobic moietyinhibits the extracellular cleavage of the transport enhancer away fromthe active biological agent, by inhibiting the attack of hydrolyticenzymes, etc. present in the extracellular tissue space, e.g. in theplasma. Thus, some preferred transport enhancers include, e.g.hydrophobic amino acids such as alanine, valine, leucine, isoleucine,methionine, proline, phenylalanine, tyrosine, and tryptophane, as wellas non-naturally occurring derivatives and analogs thereof, as mentionedsupra.

In a further option, the transport enhancer is a hydrophobic organicmoiety. Simply by way of example, the organic moiety is a C₆₋₁₈, orlarger, alkyl, aryl or heteroaryl-substituted or nonsubstituted. Theorganic moiety transport enhancer is also contemplated to encompass andinclude organic functional groups including, e.g. —C(═S) and/or —C(═O).

G. Synthesis of the Heterobifunctional Polymeric Conjugates

Synthesis of specific heterobifunctional polymer compounds is set forthin the Examples. Turning now to FIG. 1 for the purpose of illustration,one preferred method includes:

1) reacting an amine protected, activated heterobifunctional PEG polymerwith a heterobifunctional PEG polymer under basic coupling conditions toobtain a first intermediate, and

2) reacting the first intermediate with a suitable activating group suchas NHS activated ester,

3) repeating the reaction of step 1) to obtain a second intermediate,

4) deprotecting the second intermediate, and

5) reacting the activated first intermediate with the deprotected secondintermediate under coupling conditions thus achieving a high molecularweight heterobifunctional PEG conjugate.

A further method of making a polymeric conjugate according to theinvention includes:

a) reacting a compound of formula (i)

wherein:

A₁ is an activating group;

T is a protecting group;

X₁, X₃ and X₅ are independently O, S or NR₁;

R₄₄ is a polyalkylene oxide;

R₁ is selected from hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, aralkyls, and C₃₋₈ substitutedcycloalkyls;

R₄₀₋₄₁ are independently selected from the group consisting of hydrogen,C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy,phenoxy and C₁₋₆ heteroalkoxy;

n is 1 or a positive integer;

y is zero or a positive integer;

t is a positive integer; and

p is zero or one;

with a compound of the formula (ii):

wherein:

X₂, X₄ and X₆ are independently O, S or NR₁;

R_(44′) is a polyalkylene oxide;

R₁ is selected from hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cyclo-alkyls, aralkyls, C₁₋₆ substituted alkyls, and C₃₋₈ substitutedcycloalkyls;

R₄₂₋₄₃ are independently selected from the group consisting of hydrogen,C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted-cycloalkyls, aryls, substituted aryls,aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy,phenoxy and C₁₋₆ heteroalkoxy;

n′ is a positive integer;

y′ is zero or appositive integer;

t′ is a positive integer; and

p′ is zero or one;

under sufficient conditions to form a compound of formula (iii):

This method can also optionally further include the step of deprotecting(iii) to provide a useful intermediate which can be used in furthersynthesis, activated and/or conjugated to a drug, etc. Alternatively,the method can further include the step of reacting (iii) with anactivating agent under sufficient conditions to form a compound offormula (iv):

wherein A₂ is an activating group and all other variables are as definedabove.

In still further aspects, the method can include the step of convertingthe amino protecting group (T) of formula (iv) to an activating groupunder sufficient conditions to form a compound of formula (v):

wherein A₃ is an activating group.

Once a compound of formula (v) is formed, it can be reacted with abiologically active moiety, diagnostic agent or a terminal group undersufficient conditions to form a compound of formula (vi):

wherein D₂ is a biologically active moiety, diagnostic agent or terminalgroup and all other variables are as defined above.

A non-limiting list of suitable coupling agents include1,3-diisopropyl-carbodiimide (DIPC), any suitable dialkyl carbodiimide,2-halo-1-alkyl-pyridinium halides (Mukaiyama reagents),1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), propane phosphonicacid cyclic anhydride (PPACA) and phenyl dichlorophosphates, etc. whichare available, for example from commercial sources such as Sigma-AldrichChemical, or synthesized using known techniques.

Preferably the substituents are reacted in an inert solvent such astetrahydrofuran (THF), acetonitrile (CH₃CN), methylene chloride (DCM),chloroform (CHCl₃), dimethyl formamide (DMF) or mixtures thereof.Suitable bases include dimethylaminopyridine (DMAP),diisopropylethylamine, pyridine, triethylamine, KOH, potassiumt-butoxide and NaOH etc. The reactions are usually carried out at atemperature of from about 0° C. up to about 22° C. (room temperature).

More specifically, one method of forming the high molecular weightpolymer conjugates includes:

1) reacting an amine protected activated polymeric residue of theformula:

-   -   wherein n is a positive integer,

with a heterobifunctional polymeric residue of the formula:

-   -   wherein n is a positive integer,

to form a compound of the formula:

2) reacting the intermediate from step 1) with an activating group,biologically active moiety, diagnostic agent or terminal group to form acompound of the formula:

wherein D₂ is an activating group, biologically active moiety,diagnostic agent or terminal group such as, for example,ala-camptothecin:

3) deprotecting the amine portion and activating it with a moiety suchas maleimide to form a compound of the formula:

4) and thereafter, reacting the maleimide intermediate with biologicallyactive moiety such as a single chain antigen binding protein of themonoclonal antibody CC49 (which binds to TAG-72) or other fragment ofeither of the foregoing, all hereinafter designated “SCA” forconvenience, to yield an SCA immunoconjugate of the formula:

wherein n is a positive integer.

The foregoing shows an activated carbonate which yields thehetero-bifunctional with a carbamate linkage. As will be appreciated bythose of ordinary skill, an activated ester could be used the outset ofthe process to form the amide linkage.

One skilled in the art will appreciate that the conjugates preparedaccording to the methods of the present invention can increase in singleor multiple polymer units thereby resulting in repeating the same orrandom polymer subunits depending on the methods chosen to achieve thedesired conjugate.

Regardless of the route selected, some of the preferred compounds whichresult from the synthetic techniques described herein include:

wherein, B and B′ are leaving groups, activating agents, biologicallyactive agents, diagnostic agents, etc. and SCA is a singlechain-antibody.Some other preferred compounds include:

H. Methods of Treatment

Another aspect of the present invention provides methods of treatmentfor various medical conditions in mammals. The methods includeadministering to the mammal in need of such treatment, an effectiveamount of a heterobifunctional polymer composition of the invention,which has been prepared as described herein. The compositions are usefulfor, among other things, treating neoplastic disease, reducing tumorburden, preventing metastasis of neoplasms and preventing recurrences oftumor/neoplastic growths in mammals.

The amount of the compound administered will depend upon the parentmolecule, e.g. peptide, polypeptide, protein, enzyme, small moleculedrugs, etc. included therein. Generally, the amount of compound used inthe treatment methods is that amount which effectively achieves thedesired therapeutic result in mammals. Naturally, the dosages of thevarious compounds will vary somewhat depending upon the parent compound,rate of in vivo hydrolysis, molecular weight of the polymer, etc. Thoseskilled in the art will determine the optimal dosing of the compoundselected based on clinical experience and the treatment indication.Actual dosages will be apparent to the artisan without undueexperimentation.

The compounds of the present invention can be included in one or moresuitable pharmaceutical compositions for administration to mammals. Thepharmaceutical compositions may be in the form of a solution,suspension, tablet, capsule or the like, prepared according to methodswell known in the art. It is also contemplated that administration ofsuch compositions may be by the oral and/or parenteral routes dependingupon the needs of the artisan. A solution and/or suspension of thecomposition may be utilized, for example, as a carrier vehicle forinjection or infiltration of the composition by any art known methods,e.g., by intravenous, intramuscular, subdermal injection and the like.

Such administration may also be by infusion into a body space or cavity,as well as by inhalation and/or intranasal routes. In preferred aspectsof the invention, however, the compounds are parenterally administeredto mammals in need thereof.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention. The underlined and bold-faced numbers recited in theExamples correspond to those shown in the Schemes 1 to 9.

General Procedures

All reactions were ran under an atmosphere of dry nitrogen or argon.Commercial reagents were used without further purification. All PEGcompounds were dried under vacuum or by azeotropic distillation fromtoluene prior to use. NMR spectra were obtained using a Varian Mercury®300 NMR spectrometer and deuterated chloroform as the solvent unlessotherwise specified. Chemical shifts (δ) are reported in parts permillion (ppm) downfield from tetramethylsilane (TMS).

HPLC method. The reaction mixtures and the purity of intermediates andfinal products were monitored by a Beckman Coulter System Gold® HPLCinstrument employing a ZOBAX® 300 SB C-8 reversed phase column (150×4.6mm) or a Phenomenex Jupiter® 300A C18 reversed phase column (150×4.6 mm)with a multiwavelength UV detector, using a gradient of 30-90% ofacetonitrile in 0.5% trifluoroacetic acid (TFA) at a flow rate of 1mL/min.

Compound 3. A solution of 1 (0.623 g, 0.180 mmol), 2 (0.623 g, 0.180mmol), and N,N-dimethylaminopyridine (DMAP, 0.110 g, 0.90 mmol) indichloromethane (DCM, 20 mL) was stirred at room temperature for 12 hrs.The solution was washed with 0.1 N HCl (2×20 mL), dried (MgSO₄),filtered, the solvent removed under reduced pressure, and crystallizedfrom isopropyl alcohol (IPA, 25 mL) to give 3 (0.910 g, 0.134 mmol,74.3%). ¹³C NMR (67.8 MHz, CDCl₃) δ 171.91, 155.79, 155.30, 66.15,63.32, 40.34, 39.91, 34.26, 28.02.

Compound 4. A solution of 3 (0.707 g, 0.104 mmol) in DCM/trifluoroaceticacid (TFA) (8 mL:4 mL) was stirred at room temperature for 3.5 hrs atroom temperature. The solvent was removed under reduced pressure and theresulting solid washed with either to yield 4 (0.707 g,). 104 mmol,˜100%). ¹³C NMR (67.8 MHz, CDCl₃) δ 172.18, 155.94, 66.62, 66.32, 63.49,40.49, 39.71, 34.46.

Compound 5. To a solution of 3 (0.910 g, 0.134 mmol),2-mercaptothiazoline (2-MT, 0.0319 g, 0.268 mmol), and DMAP (0.032.7 g,0.268 mmol) in DCM (15 mL) cooled at 0° C. for 15 min was added1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDC,0.0513 g, 0.268 mmol) and the reaction solution allowed to graduallywarm to room temperature and then stirred for 12 hrs. The PEG derivativewas precipitated with ethyl ether, collected by filtration, andcrystallized from IPA (19 mL) to give 5 (0.820 g, 0.121 mmol, 90.0%).¹³C NMR (67.8 MHz, CDCl₃) δ 200.94, 171.73, 155.85, 155.36, 65.75,63.37, 55.54, 40.37, 39.94, 38.73, 28.08.

Compound 6. To a solution of 4 (0.668 g, 0.098 mmol) in DCM (15 mL) wasadded DMAP to adjust the pH to 7.0. Compound 5 (0.677 g, 0.098 mmol) wasadded and the reaction mixture stirred at room temperature for 12 hrs.The solution was washed with 0.1 N HCl (2×20 mL), dried (MgSO₄),filtered, solvent removed under reduced the pressure and residuecrystallized from isopropyl alcohol (IPA, 25 mL) to give 6 (1.053 g,0.077 mmol, 79.0%). ¹³C NMR (67.8 MHz, CDCl₃) δ 171.97, 170.72, 155.87,155.36, 66.83, 66.24, 63.39, 40.40, 39.99, 38.74, 36.50, 34.34, 28.08.

Compound 8. To a solution of 6 (0.616 g, 0.045 mmol),20-(S)-camptothecin alaninate trifluoroacetic acid salt (0.0706 g, 0.136mmol), and DMAP (0.111 g, 0.906 mmol) in DCM (10 mL) cooled at 0° C. for15 min was added EDC (0.026 g, 0.136 mmol) and the reaction solutionallowed to warm to room temperature. After stirring for 12 hrs, thesolution was washed with 0.1 N HCl (2×20 mL), dried (MgSO₄), filtered,the solvent removed under reduced pressure, and the residue crystallizedfrom isopropyl alcohol (IPA, 13 mL) to give 8 (0.536 g, 0.038 mmol,85.0%). ¹³C NMR (67.8 MHz, CDCl₃) δ 171.09, 170.83, 170.63, 166.48,156.82, 155.99, 151.82, 148.46, 146.01, 144.98, 130.77, 130.12, 129.40,128.06, 127.77, 127.58, 119.72, 95.58, 66.97, 66.77, 63.57, 49.74,47.56, 40.55, 40.14, 38, 90, 36.70, 36.41, 31.48, 28.22, 17.58, 7.40.

Compound 9. A solution of 8 (0.536 g, 0.038 mmol) in DCM/TFA (8 mL:4 mL)was stirred at room temperature for 2 hrs. The solvent was removed underreduced pressure and the residue washed with ethyl ether to give 9(0.536 g, 0.038 mmol, ˜100%). ¹³C NMR (67.8 MHz, CDCl₃) δ 170.99,170.81, 170.60, 166.25, 156.58, 155.79, 151.56, 148.19, 145.79, 144.79,130.71, 129.92, 129.12, 127.91, 127.63, 127.37, 119.46, 95.44, 66.71,66.54, 63.34, 49.59, 47.45, 40.34, 39.59, 38, 78, 36.34, 36.08, 31.24,17.24, 7.20.

Compound 11. To a solution of 9 (0.818 g, 0.059 mmol) in DCM (15 mL) wasadded DMAP to adjust the pH to 7.0, then 10 was added and the solutioncooled to 0° C. 1,3-diisopropylcarbodiimide (DIPC, 0.0554 μL, 0.354mmol) was added to the reaction and the mixture allowed to warm to roomtemperature with stirring for 12 hrs. The solution was washed with 0.1 NHCl (2×20 mL), dried (MgSO₄), filtered, the solvent removed underreduced pressure and the residue crystallized from isopropyl alcohol(IPA, 16 mL) to give 11 (0.65 g, 0.046 mmol, 78%). ¹³C NMR (67.8 MHz,CDCl₃) δ 172.22, 171.10, 170.84, 170.60, 170.32, 166.48, 156.83, 155.99,151.82, 148.48, 146.04, 144.98, 133.70, 130.76, 130.13, 129.43, 128.06,127.78, 127.60, 119.75, 95.56, 66.99, 66.77, 63.58, 49.74, 47.56, 40.57,38.92, 37.39, 36.72, 36.43, 36.05, 31.48, 28.08, 26.17, 25.18, 24.86,17.30, 7.40.

Compound 12. A. Reduction of protein CC49: to a solution of 28 mg (2.79mg/ml) of CC49 in 100 mM sodium phosphate, pH 7.8, at 37° C., 2 mM EDTAwas added with 2 mM DTT and reaction allowed to proceeded for 2 hrs. TheDTT was removed by a desalting column equilibrated with a solution of100 mM sodium phosphate, pH 6.5, and 2 mM EDTA. The final concentrationof the reduced protein was 0.39 mg/ml (˜23 mg, ˜60 ml, 83%).

B. PEGylation: CC49 and 11 were mixed at 1:10 molar ratio in a solutionof 100 mM sodium phosphate, pH 6.5, 2 mM EDTA and reacted at 25° C. for2 hrs.

C. Purification of CC49-PEG-CPT: the pH value of the reaction solutionwas adjusted to 5 with HOAc and water (˜200 mL) was added to reduce theconductivity of the solution to less than 2 mS and the mixture loadedonto a Poros HS column at 5 mL/min. The product was eluted by 1 M NaClin 10 mM sodium phosphate solution and the fractions of protein peakwere combined and concentrated using a 30 k Centriplus centrifuge tube.The concentrated sample was dialyzed against saline and analyzed foractive component. An iodine stain test found no non-protein conjugatedPEG species in the product.

Compound 13. A solution of 6 (4.50 g, 0.335 mmol) in DCM/TFA (30 mL: 15mL) was stirred at room temperature for 3.5 hrs at room temperature. Thesolvent was then removed under reduced pressure and the resulting solidwashed with ether to yield 13 (4.30 g, 0.320 mmol, 95.6%). ¹³C NMR (67.8MHz, CDCl₃) δ 171.59, 155.58, 66.36, 65.89, 63.02, 40.05, 39.36, 38.61,35.85, 33.96.

Compound 14. To a solution of 13 (4.30 g, 0.320 mmol) in DCM (50 mL) wasadded DMAP to pH 7.0. Then compound 5 (2.20 g, 0.320 mmol) was added andthe reaction mixture stirred at room temperature for 12 hrs. Thesolution was washed with 0.1 N HCl (2×30 mL), dried (MgSO₄), filtered,the solvent removed under reduced pressure and the residue crystallizedfrom isopropyl alcohol (IPA, 25 mL) to give 14 (5.30 g, 0.260 mmol,81.2%). ¹³C NMR (67.8 MHz, CDCl₃) δ 171.77, 170.60, 155.75, 66.73,66.12, 63.28, 40.29, 39.86, 38.66, 36.41, 34.19, 27.99.

Compound 15. A solution of 14 (5.30 g, 0.260 mmol) in DCM/TFA (30 mL:15mL) was stirred at room temperature for 3.5 hrs at room temperature. Thesolvent was then removed under reduced pressure and the resulting solidwashed with ether to yield 15 (5.30 g, 0.260 mmol, ˜100%). ¹³C NMR (67.8MHz, CDCl₃) δ 171.77, 170.60, 155.75, 66.73, 66.12, 63.28, 40.29, 39.86,38.66, 36.41, 34.19.

Compound 18. To a solution of Rhodamine B base (1.00 g, 2.09 mmol),Glycine t-butylester hydrochloride salt (0.670 g, 4.0 mmol) and DMAP(0.767 g, 8.0 mmol) in DCM (30 mL) cooled to 0° C. for 15 min was addedEDC (0.767 g, 4.0 mmol). The reaction mixture was allowed to warm toroom temperature and stirred for 12 hrs. The solution was washed with0.1 N HCl (2×30 mL), dried (MgSO₄), filtered, solvent removed underreduced pressure and the residue purified by silica gel columnchromatography using hexane and ethyl acetate (3:2, v/v) as elutingsolvents to give 18 (0.937 g, 1.58 mmol, 76%). ¹³C NMR (67.8 MHz, CDCl₃)δ 167.30, 166.73, 153.06, 153.01, 148.36, 132.05, 130.60, 129.31,127.63, 123.46, 122.67, 107.64, 104.69, 97.21, 80.80, 64.74, 44.10,42.03, 27.63, 12.41.

Compound 19. A solution of 18 (0.937 g, 1.58 mmol) in DCM/TFA (16 mL:8mL) was stirred at room temperature for 2 hrs. The solvent was removedunder reduced pressure and the residue washed by ethyl ether to give 19(0.930 g, 1.57 mmol, ˜100%).

¹³C NMR (67.8 MHz, CDCl₃) δ 169.30, 167.83, 152.72, 152.15, 144.14,133.09, 130.28, 128.83, 123.64, 123.41, 112.32, 103.89, 64.69, 48.47,41.49, 11.44.

Compound 20. To a solution of 19 (0.421 g, 0.785 mmol), 2-MT (0.140 g,1.18 mmol), and DMAP (0.287 g, 2.30 mmol) in DCM (15 mL) cooled at 0° C.for 15 min was added EDC (0.226 g, 1.18 mmol) and the reaction solutionallowed to gradually warm to room temperature and then stirred for 12hrs. The solution was washed with 0.1 N HCl (2×20 mL), dried (MgSO₄),filtered, the solvent removed under reduced pressure to give 20 (0.450g, 0.706 mmol, 90%). ¹³C NMR (67.8 MHz, CDCl₃) δ 200.68, 168.90, 167.73,153.27, 152.07, 148.10, 139.66, 132.47, 130.20, 129.39, 127.94, 123.63,122.85, 108.19, 98.14, 65.11, 55.77, 51.31, 45.68, 44.68, 33.67, 29.09,12.53.

Compound 21. To a solution of 15 (2.7 g, 0.134 mmol) in DCM was addedDMAP to adjust the pH to 7. Compound 20 (171 mg, 0.268 mmol) was addedand the reaction solution was stirred at room temperature for 12 hrs.The reaction mixture was washed with 0.1N HCl, the solvent evaporatedunder reduced pressure, and the solid crystallized from IPA to yield 21(2.3 g, 0.112 mmol, 84%). ¹³C NMR (67.8 MHz, CDCl₃) δ 201.00, 170.78,167.73, 155.88, 152.86, 148.80, 132.51, 129.86, 128.06, 127.87, 123.59,122.53, 108.14, 98.09, 66.86, 63.45, 55.57, 44.37, 43.99, 40.43, 38.80,38.54, 36.57, 34.32, 28.10, 12.18.

Compound 22. To a solution of 21 (2.3 g, 0.112 mmol), 2-MT (0.027 g,0.224 mmol), and DMAP (0.027 g, 0.224 mmol) in DCM (1, mL) cooled at 0 Gwas added EDC (0.043 g, 0.224 mmol). The reaction solution was graduallywarmed to room temperature and stirred for 12 hrs. The PEG derivativewas precipitated with ethyl ether, filtered, and crystallized from IPAto give 22 (2.0 g, 0.097 mmol, 86%). ¹³C NMR (67.8 MHz, CDCl₃) δ 200.00,171.70, 170.64, 167.96, 167.68, 155.79, 152.86, 148.24, 132.38, 129.86,127.84, 127.72, 123.55, 122.38, 104.12, 97.47, 66.80, 63.37, 55.51,44.91, 40.37, 38.72, 38.44, 36.50, 28.08, 12.26.

Compound 23. A solution of 22 (2.0 g, 0.097 mmol),3,5-dimethyl-4-hydroxybenzyl alcohol (0.059 g, 0.388 mmol), and DMAP(0.048 g, 0.388 mmol) in DCM (10 mL) was refluxed for 12 hrs. The PEGderivative was precipitated with ethyl ether, filtered, and crystallizedfrom IPA to give 23 (1.9 g, 0.096 mmol, 99%). δ 170.40, 168.40, 167.64,167.38, 155.59, 152.65, 148.01, 132.13, 129.66, 129.11, 127.66, 126.16,123.31, 122.14, 107.48, 103.99, 97.26, 66.88, 63.34, 43.70, 40.15,38.52, 38.25, 36.26, 34.37, 15.76, 12.07.

Compound 24. To a solution of 23 (1.9 g, 0.097 mmol) andN,N′-disuccinimidyl carbonate (0.199 g, 0.775 mmol) in DCM (20 mL) andDMF (2 mL) cooled to 0° C. was added pyridine (0.063 μL, 0.775 mol). Thereaction solution was gradually warmed to room temperature and stirredfor 12 hrs. The PEG derivative was precipitated with ethyl ether,filtered, and crystallized from IPA to give 24 (1.58 g, 0.075 mmol,77%).

¹³C NMR (67.8 MHz, CDCl₃) δ 171.22, 170.49, 168.35, 168.05, 167.73,167.47, 155.65, 152.71, 148.07, 132.24, 129.69, 128.09, 127.72, 123.39,122.23, 107.53, 104.02, 97.30, 66.65, 63.19, 43.77, 40.22, 38.57, 38.31,36.22, 34.43, 24.89, 15.79, 12.13.

Compound 25. Activated PEG linker 24 was added to a solution of GFP (2mg/ml) in 0.05 M HEPES, pH 7.8, with a molar ratio of 30:1 (PEG:GFP).The solution was stirred at 25° C. under N₂ for 45 min, the pH of thesolution was lowered by adding sodium phosphate buffer, pH 6.4, to afinal concentration of 50 mM. The free PEG was removed on a Superdex 200Hiload 16/60 column (Amersham Pharmacia Biotech, Piscataway, N.J.) usinga Biocad Perfusion Chromatography Workstation. The elution buffer wascomprised of 10 mM sodium phosphate, pH 6.8 and 150 mN NaCl. Thefractions that exhibited both absorbance: at 280 nm and fluorescencewere pooled and concentrated using ultrafree-15 centrifugal filterdevice with 30 k NMWL membrane (Millipore Corp., Bedford, Mass.). ThePEG-GFP (25) concentration was determined by UV at 489 nm using anextinction coefficient of 55,000 cm⁻¹ M⁻¹.

Compound 27. To a solution of 6, 26, and DMAP in DCM is added EDC andthe solution stirred at room temperature for 12 hrs. The solvent isremoved under reduced pressure and the solid crystallized from IPA togive 27. The structure of 27 is confirmed by ¹³C NMR.

Compound 28. A solution of 27 in DCM/TFA is stirred at room temperaturefor 12 hrs. The solvent is removed under reduced pressure and the solidcrystallized from IPA to give 28. The structure of 28 is confirmed by¹³C NMR.

Compound 29. To a solution of 10, 2-MT, and DMAP in DCM cooled at 0° C.for 15 min is added EDC and the reaction solution allowed to graduallywarm to room temperature and then stirred for 2 hrs. The solution isthen washed by 0.1 N HCl, dried (MgSO4), and the solvent removed underreduced pressure to give 29. The structure of 29 is confirmed by ¹³CNMR.

Compound 30. A solution of 28, 29 and DMAP in DCM is stirred at roomtemperature for 12 hrs. The solvent is removed under reduced pressureand the solid crystallized from IPA to give 30. The structure of 30 isconfirmed by ¹³C NMR.

Compound 31. To a solution of 30, 7, and DMAP in DCM cooled at 0° C. for15 min is added EDC and the reaction solution allowed to warm to roomtemperature. After stirring for 12 hrs, the solution is washed with 0.1N HCl, dried (MgSO₄), filtered, the solvent removed under reducedpressure, and the residue crystallized from IPA to give 31. Thestructure of 31 is confirmed by ¹³C NMR.

What we claim:
 1. A compound of the formula:

wherein: X₁-X₆ are independently O, S or NR₁; R₄₄ and R_(44′) areindependently selected polyalkylene oxides; R₁ is selected from thegroup consisting of hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈cycloalkyls, C₁₋₆ substituted alkyls, aralkyls, and C₃₋₈ substitutedcycloalkyls; R₄₀₋₄₃ are independently selected from the group consistingof hydrogen, C₁₋₆ alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆substituted alkyls, C₃₋₈ substituted cycloalkyls, aryls, substitutedaryls, aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆alkoxy, phenoxy and C₁₋₆ heteroalkoxy; y and y′ are independently zeroor a positive integer; p and p′ are independently zero or one n and n′are independently a positive integer; a and b are independently zero ora positive integer, provided that a+b is greater than or equal to 2; zis a positive integer; D₁₀ and D₁₁ are independently selected from thegroup consisting of OH, halogens, drugs, enzymes, proteins,therapeutically active compounds, dyes, chelating agents', isotopelabeled compounds; Y₇₋₉ are independently selected from the groupconsisting of O, S NR_(1″); R_(1″) is hydrogen or methyl; R₉₋₁₈ areindependently selected from the group consisting of hydrogen, C₁₋₆alkyls, C₃₋₁₂ branched alkyls, C₃₋₈ cycloalkyls, C₁₋₆ substitutedalkyls, C₃₋₈ substituted cycloalkyls, aryls, substituted aryls,aralkyls, C₁₋₆ heteroalkyls, substituted C₁₋₆ heteroalkyls, C₁₋₆ alkoxy,phenoxy and C₁₋₆ heteroalkoxy; L₃₋₄ are independently selectedbifunctional linkers; Q is selected from the group consisting of-L₅-C(═Y₁₀)— wherein L₅ is the same group that defines L₃₋₄ and Y₁₀ isthe same group that defines Y₇₋₉, hydrophobic moieties, bifunctionallinking moieties and combinations thereof; l, k, m and o areindependently positive integers; j and h are independently zero or apositive integer; g, i and q are independently zero or one; and B′ isselected from the group consisting of leaving groups, activating groups,OH, biologically active moieties and diagnostic agents.
 2. The compoundof claim 1, having the formula


3. The compound of claim 2, selected from the group consisting of:

wherein, B′ is selected from the group consisting of leaving groups,activating agents, OH, biologically active agents, and diagnosticagents.
 4. The compound of claim 3, wherein B′ is selected from thegroup consisting of maleimide and residues of hydroxyl-containing oramine-containing compounds, wherein there is at least one hydroxyl oramine available in the hydroxyl- or amine-containing compounds which canreact and link with the polymeric conjugate.
 5. The compound of claim 3,wherein B′ is selected from the group consisting of anthracyclines,daunorubicin, doxorubicin, p-hydroxyaniline mustard, cytosine, ara-C,gemcitibine, camptothecin, vancomycin, paullones, paclitaxel, cisplatin,vincristine and vinblastine.